Use of sedoheptulose for prevention or treatment of inflammation

ABSTRACT

The invention discloses sedoheptulose for use in the prevention or treatment of inflammation.

The technical field of the present invention is the prevention ortreatment of inflammation.

Inflammation is part of the complex biological response of vasculartissues to harmful stimuli, such as pathogens, damaged cells, orirritants. The classical signs of acute inflammation are pain, heat,redness, swelling and loss of function. Inflammation is a protectiveattempt by the organism to remove the injurious stimuli and to initiatethe healing process. Inflammation is a stereotyped response, andtherefore it is considered as a mechanism of innate immunity, ascompared to adaptive immunity, which is specific for each pathogen.

Inflammation can be classified as either acute or chronic. Acuteinflammation is the initial response of the body to harmful stimuli andis achieved by the increased movement of plasma and leukocytes(especially granulocytes) from the blood into the injured tissues. Acascade of biochemical events propagates and matures the inflammatoryresponse, involving the local vascular system, the immune system andvarious cells within the injured tissue. Prolonged inflammation, knownas chronic inflammation, leads to a progressive shift in the type ofcells present at the site of inflammation and is characterized bysimultaneous destruction and healing of the tissue from the inflammatoryprocess.

Inflammatory abnormalities (inflammatory disorders) are a large group ofdisorders which underlie a vast variety of human diseases. The immunesystem is often involved with inflammatory disorders, demonstrated inboth allergic reactions and some myopathies, with many immune systemdisorders resulting in abnormal inflammation. Non-immune diseases withetiological origins in inflammatory processes include cancer,atherosclerosis and ischaemic heart disease. Examples of disordersassociated with inflammation include acne vulgaris, asthma, autoimmunediseases, celiac disease, chronic prostatitis, glomerulonephritis,hypersensitivities, inflammatory bowel diseases, pelvic inflammatorydisease, reperfusion injury, rheumatoid arthritis, sarcoidosis,transplant rejection, vasculitis, interstitial cystitis,atherosclerosis, allergy, myopathies, leucocyte defects and cancer.

Medical treatment of acute and chronic inflammation has been performedwith corticosteroids or non-steroidal anti-inflammatory drugs (NSAIDs).

Also anti-inflammatory foods have been suggested to treat or preventinflammation. Prostaglandins are hormone-like substances that affect thebody in variety of ways, also regulating inflammatory mediation. Ananti-inflammatory diet includes less food that createinflammation-causing prostaglandins (PGE2) in the body, and more foodsthat create anti-inflammatory prostaglandins (PGE1 and PGE3). Suggesteddiets to prevent inflammation include those rich in vegetables and lowin simple carbohydrates and fats, such as saturated fats and trans-fats.Anti-inflammatory foods include most colourful fruits and vegetables,oily fish (which contain higher levels of omega-3 fatty acids), nuts,seeds and certain spices, such as ginger. Extra-virgin olive oilcontains the chemical oleocanthal that acts similarly to ibuprofen.Omega-3 fatty acids have been shown to disrupt inflammation cellsignaling pathways by binding to the GPR120 receptor.

However, there is still a need for prevention and treatment ofinflammation, especially chronic inflammation. Such prevention andtreatment should be provided without severe side effects or, preferably,without the risk for long term adverse developments, such as addiction,metabolic disorders, declining efficacy, etc.

Therefore, the present invention provides sedoheptulose for use in theprevention or treatment of inflammation.

Sedoheptulose (CAS number: 3019-74-7; PubChem: 5459879; ChemSpider:4573620; MeSH: sedoheptulose; ChEBI: CHEBI:16802) or D-altro-heptuloseis a ketoheptose—a monosaccharide with seven carbon atoms and a ketonefunctional group. It is one of the few heptoses found in nature.Sedoheptulose is producible by extraction from natural sources or bychemical synthesis. It can be purified to high purity degrees (e.g. >60%purity, preferably >85% purity, more preferred >90% purity, morepreferred >95% purity, even more preferred >99% purity,especially >99.9% purity).

Sedoheptulose is a relatively unknown metabolite compared to itsphosphorylated form, sedoheptulose-7-phosphate (S7P), which isrecognised as an intermediate molecule of primary glucose metabolism.The natural existence of sedoheptulose, a ketoheptose, was firstreported in plants and was subsequently identified in human urine, bloodspots and recently, within mouse cells. S7P is derived from thetransketolase-catalysed conversion of ribose-5-phosphate (R5P) andxylulose-5-phosphate (X5P). This reaction occurs in the non-oxidativearm of the pentose phosphate pathway and generatesglyceraldehyde-3-phosphate (G3P), a key glycolytic intermediate, inaddition to S7P. G3P and S7P are also produced by transaldolase usingfructose-6-phosphate (F6P) and erythrose-4-phosphate (E4P) assubstrates. S7P is thus a crucial intermediate in the non-oxidative PPPand S7P bioavailability therefore contributes to cellular carbon flux.Recently, data from several groups have indicated that sedoheptulosekinase may also produce S7P by direct phosphorylation of sedoheptulose(Haschemi et al., Cell Metab. 15 (2012), 813-826). This findingdemonstrated the unexpected existence of an additional carbon source,sedoheptulose, which actively participates in cellular carbonmetabolism.

Free sedoheptulose can either be diet-derived from fruits and vegetablesor formed enzymatically by the dephosphorylation of endogenouslyproduced S7P. Thus far, no specific sedoheptulose transporter orS7P-specific phosphatase has been reported. Other bio-active heptosesinclude the phenolic compound 7-O-Galloyl-sedoheptulose (GS),mannoheptulose and glucoheptose. GS was reported to be protective indiabetic injury of the kidney by alleviating inflammatory responses.Mannoheptulose (various patents pending) is an isomer of sedoheptuloseand a potent hexokinase inhibitor commonly found in avocados.Glycoheptose, although the chemical nature of this compound remainsunidentified, has been shown to serve as accessible carbohydrate sourcein rabbits. Sedoheptulosan is the anhydride of sedoheptulose and mightalso serve as source for free sedoheptulose.

Free sedoheptulose can be isolated, e.g. from the plant sedum spectabileand has been previously reported to contain high amounts ofheptose-carbohydrates.

Sedoheptulose kinase phosphorylates free sedoheptulose, which enablescells to directly route sedoheptulose to carbohydrate metabolism,similar to hexokinase and glucose. According to the present invention,sedoheptulose is a direct carbon source that feeds primary carbohydratecatabolism and anabolism, whereupon CARKL constitutes its entry point.Sedoheptulose can therefore surprisingly be compared to glucose andfructose because these compounds all exist as free carbohydrates andphosphorylated forms. To enter metabolism, free carbohydrates areenergetically activated by an initial phosphorylation event. Hexokinasephosphorylates glucose to form G6P and is a key determinant of cellularglucose flux. Fructose is phosphorylated by ketohexokinase to formfructose-1-phosphate (F1P), which must first be converted by aldolase toglyceraldehyde and dihydroxyacetone phosphate (DHAP). DHAP can thendirectly enter the carbon-cycle, whereas glyceraldehyde must be furtherphosphorylated. The formation of S7P from sedoheptulose by sedoheptulosekinase, in a manner analogous to G6P formation from glucose byhexokinase, enables free sedoheptulose to readily enter the catabolicsystem. It was also reported that F6P phosphorylation is competitivelyinhibited in the presence of high S7P concentrations. Interestingly,fractions containing fructose 1,6-bisphosphatase (FBPase) were alsoreported to possess sedoheptulose 1,7-bisphosphatase (SBPase) activity.

The present invention therefore is also based on a co-existence of thehexose- and heptose-(bis)phosphate shunts. Notably, high glucose levelsor incubation with F2,6bP augmented the S1,7bP formation in perfused ratliver and in rat liver cytosol, respectively. Glucagon, in contrast tohigh glucose, favoured S1,7bP dephosphorylation and therefore S7Pformation. These results show that sedoheptulose metabolism is sensitiveto hormonal control.

Sedoheptulose metabolism participates in metabolic regulation; in fact,sedoheptulose directs metabolic fluxes by providing an S7P supplyindependently from glucose. Increased CARKL expression (and thereforeincreased sedoheptulose consumption) in a mouse macrophage cell lineresulted in reduced G3P, X5P and R5P steady state levels, whereas CARKLknockdown showed the reverse effect. Notably, the basal sedoheptuloselevels were not changed by CARKL perturbation. The S7P levels were notchanged by overexpression but decreased significantly by CARKL loss,indicating sedoheptulose phosphorylation as a rate-limiting factor forglycolysis-derived G3P distribution. Therefore, the regulation of S7Pavailability might be the mechanism by which CARKL or excessive amountsof sedoheptulose modulate cellular metabolism.

The present invention is therefore directed to use of C7, e.g. tobalance C6-(over)consumption, obesity, diabetes, immune functionand—generally—vertebrate's energy utilization. The C7 supplementtherefore also provides an effective strategy to manage physiologicalredox-regulation and thereby also related disorders. With the presentinvention, it turned out that, both, high sedoheptulose and highsedoheptulose kinase results in enhanced sedoheptulose turn-over.Moreover, tissue expression of sedoheptulose kinase reveals thatmetabolic organs such as liver or adipose-tissue have the capability toconsume sedoheptulose. Additionally, brown-adipose tissue (burns highamounts of lipids) express significantly more (˜2.5×) sedoheptulosekinase than white-adipose tissue.

Sedoheptulose (and/or other heptoses), administered to patients havinginflammation or being at risk of suffering from inflammation, minimizesthe glycemic-load and positively contributes to prevent e.g.diet-induced obesity and/or diabetes. Furthermore, data from transgenicsedoheptulose kinase animals—as model for enhanced sedoheptulosemetabolism—show that high sedoheptulose metabolism increaseslipid-oxidation (indicated by lower RQ values). This is a beneficialeffect of enhanced sedoheptulose turnover. Also energy expenditure islower in animals with high sedoheptulose turnover. Furthermore, itappears that CARKL mice are more insulin sensitive than control animals.Taken together, this again shows that sedoheptulose metabolism is aneffective mean to regulate the metabolic-phenotype in vertebrates.

With the present invention, specific and surprising anti-inflammatoryeffects could be shown to be caused by high sedoheptulose turnover:Enhanced inflammation plays an important role in the development ofdiseases, such as obesity related disorders like insulin resistance. Ina functional kinase screen for novel regulators of macrophagesactivation, sedoheptulose kinase was found to repress thelipopolysaccharide (LPS) induced tumour necrosis factor α secretion. Inthe same screen, overexpression of hexokinase, ketohexokinase andphosphofructokinase (high glucose/fructose metabolism) had the oppositeeffect. This showed opposite effects of hexose and heptose-carbohydratemetabolism: heptose inhibit inflammation. Moreover, increased CARKLexpression repressed LPS-induced macrophage activation and resulted inblunted intracellular superoxide formation, whereas loss of CARKL(mimics reduced sedoheptulose metabolism) enhanced the inflammatoryresponse of those cells.

In addition to these effects, it was shown in the course of establishingthe present invention that cytokine production is decreased afterinflammation (elicited by LPS injections) in CARKL overexpressinganimals compared to wild-type controls as well as in adipose tissue ofCARKL mice, or by sedoheptulose supplementation of the latter tissueex-vivo Together, this showed that administration of sedoheptulosedampens inflammation and therefore positively affects healthcare-relatedaspects of patients having or being at risk for inflammatory diseases,especially chronic inflammatory diseases. Chronic inflammatory diseasesaccording to the present invention are defined as persistentinflammation, local or systemic, of any etiology, known (like forinstance persistent infection) or currently unknown (like for instancesarcoidosis or most cases of chronic rhinosinusitis).

Preferred inflammations to be prevented or treated according to thepresent invention are systemic inflammatory response syndrome (SIRS),sepsis, meta-inflammation, inflammaging, inflammation in thepathogenesis of an inflammation-related disorder, inflammation in thepathogenesis and inflammation in the course of acute or chronictransplant rejection. Accordingly, a preferred inflammation according tothe present invention is wherein the inflammation is SIRS or sepsis,defined as massive systemic inflammation, especially in response topolytrauma or infection, including all the organ-specificmanifestations, especially acute respiratory distress syndrome (ARDS) ormultiple organ dysfunction syndrome (MODS).

Another preferred inflammation according to the present invention iswherein the inflammation is meta-inflammation or inflammaging, definedas chronic (low grade) systemic inflammation as reflected by increasedlevels of pro-inflammatory cytokines (like for instance tumor necrosisfactor alpha or interleukin-6) or other inflammation markers (like forinstance c-reactive protein or serum amyloid alpha) in peripheral bloodand involved in aging, adiposity, atherosclerosis, impaired glucosetolerance and type 2 diabetes, neurodegenerative disorders, likedementia, major depressive disorder (MDD) and cancer.

Another preferred inflammation according to the present invention iswherein the inflammation is involved, causally or otherwise, in thepathogenesis of an inflammation-related disorder, especially adiposity,impaired glucose tolerance, diabetes mellitus and diabeticcomplications, metabolic syndrome, atherosclerosis, inflammatory boweldisease (Crohn's disease, colitis ulcerosa and rarer entities), chronicinflammatory liver disease including chronic viral hepatitis, autoimmunehepatitis, primary sclerosing cholangitis and fatty liver disease in allstages, chronic obstructive pulmonary disease (COPD) and asthmabronciale, inflammatory diseases of the central nervous system includingmultiple sclerosis, Alzheimer's and Parkinson's disease or peripheralnervous system including diabetic polyneuropathy, Guillain-Barre andrelated syndromes and neuritides/neuropathies associated with autoimmunecollagen tissue diseases or chronic infection like for instance Lymedisease, osteoporosis, osteoarthritis, arthritides like rheumatoidarthritis or ankylosing spondylitis and other rheumatic disordersincluding the entire spectrum of autoimmune connective tissue disordersand myositis, vasculitis of any etiology and manifestation, age-relatedmacular degeneration (AMD) chronic inflammatory skin disorders likepsoriasis and atopic dermatitis, periodontitis, major depressivedisorder and cancer.

Another preferred inflammation according to the present invention iswherein the inflammation is involved in the pathogenesis of cancerincluding cancer causation, progression, metastasis, resistance totherapy, relapse after therapy and cancer manifestations, especiallycancer-related cachexia, anemia, fatigue and depression. Given theuniversal involvement of inflammation in the pathogenesis of cancer thepresent invention is applicable to all cancer entities regardless oftissue origin, differentiation or localization including all types ofcarcinoma and sarcoma, melanoma and other tumors of neural crest-derivedcells, tumors of the central nervous system, leukemia and lymphoma.

Another preferred inflammation according to the present invention iswherein the inflammation is involved in acute or chronic transplantrejection, especially rejection of allogeneic lung, liver, kidney,heart, intestine or pancreatic islet cell transplants or, conversely, inacute or chronic graft-versus-host disease following autologous,syngeneic or allogeneic bone marrow, peripheral blood stem cell (PBSC)or cord blood transplantation.

Other preferred inflammations according to the present invention isselected from the group consisting of an inflammation of adipose tissue,an inflammation of the nervous system (neuroinflammation), aninflammation of blood vessels (vascular inflammation) and aninflammation of the peritoneum especially as a consequence of peritonealdialysis as used for kidney replacement therapy.

On the other hand, the present invention also relates to the preventionand treatment of all kind of sedoheptulose deficits, i.e. to treat asedoheptulose deficit so that disorders that are due to suchsedoheptulose deficits can be prevented. In order to treat suchsedoheptulose deficits, a sedoheptulose containing composition can beadministered to a patient having or being at risk of developing suchsedoheptulose deficit (or sedoheptulose deficit syndrome), e.g. asnutritional supplement or in combination with food and/or beverages.

Sedoheptulose is not toxic; it is a natural source of carbohydrates forhuman metabolism. It may therefore be administered in virtually allmedically reasonable ways without risking unexpected side effects oradverse reactions. According to preferred administration dosages,especially single dosage forms, of the present invention, sedoheptuloseis administered in an amount of 0.001 to 300 g per daily dosage,preferably in an amount of 0.5 to 200 g per daily dosage, especially inan amount of 1 to 100 g per daily dosage.

Preferred administration of sedoheptulose is performed parentally,subcutaneously, by intravenous, intratumoural subcutaneous,intramuscular or intraperitoneal injection, especially whereinsedoheptulose is administered orally.

Sedoheptulose may virtually be included in almost any pharmaceuticalformulation form, including pharmaceutically acceptable excipients.Preferably, sedoheptulose is administered as a powder, granulate,tablet, pellet, capsule or syrup containing sedoheptulose.

According to a preferred embodiment, sedoheptulose is administered in anamount of 0.001 to 300 g, preferably in an amount of 0.5 to 200 g,especially in an amount of 1 to 100 g. These administration dosages aree.g. for a normal 70 to 80 kg human individual but can be adapted easilybased on sex, inflammation degree (or risk), other therapeutic regimen,physiological status/fitness of the patient, etc.

According to a preferred embodiment, sedoheptulose is administered incombination with hexokinase inhibitors, preferably mannoheptulose or2-deoxy-D-glucose. Hexokinase inhibitors are well known in the art andcan be applied in the present invention in the amounts and dosagesalready known or suggested in the prior art to the patient. For example,the inhibitor can be added in an amount of 0.0001% to 50% w/w,especially 0.001% to 1%, relative to the sedoheptulose content (e.g.0.0001 to 50 mg, if 100 mg sedoheptulose is administered).

According to another embodiment, the present invention relates to amethod for storage of human organs for transplantation purposes whereinthe organ to be transplanted to a patient is stored in a storagesolution comprising sedoheptulose. This aspect is based on theanti-inflammatory capacity of sedoheptulose and serves the same aim ofreducing inflammation in the transplant patient (to whom sedoheptulosemay be applied as well): The anti-inflammatory property of thesedoheptulose storage solution that is used for impregnating the organto be transplanted also safeguards the reduction or prevention ofinflammatory processes in the isolated organ itself during storage andalso after transplantation so that not only anti-inflammatory protectionis present in the patient, but also in the organ that has beentransplanted.

In a preferred embodiment of this method, the storage solution comprisessedoheptulose in a concentration of 0.001 to 500 mg/ml, preferably in aconcentration of 0.01 to 300 mg/ml, especially in a concentration of 0.1to 150 mg/ml, defined as the concentration of all liquid materialattached to or covering the organ to be transplanted (i.e. the solutionthat remains in the organ storage containment after removing the organfrom the containment).

According to another aspect, the present invention also relates to aperitoneal dialysis solution containing sedoheptulose in a concentrationof 0.001 to 500 mg/ml, preferably in a concentration of 0.01 to 300mg/ml, especially in a concentration of 0.1 to 150 mg/ml.

The present invention is further described in the following examples andthe figures, yet without being restricted thereto.

FIG. 1: (A) Pooled samples from carrots or leaves of indoor grown SedumSpectabile plants were analyzed by gas-chromatography coupled to massspectrometry to specifically measure relative glucose (C6) andsedoheptulose (C7) levels. The ratio of C6/C7 was calculated by dividingtheir respective peak area. (B) Sedoheptulose and glucose levels werefurther assessed by GC-MS in serum of individuals before and after fooduptake to investigate if diet-derived C7 can enter the humanblood-stream. Sedoheptulose and glucose level of four overnight fasted(fasted) individuals (empty bars) were compared to levels measured twohours after a meal (diet-fed, grey bars) containing mainly carrots (˜700g) with some olive-oil, salt and pepper. The change in serumcarbohydrate levels was expressed as mean fold change (n=4, +/−S.D.).(C) Concentrations of glucose, fructose and sedoheptulose were measuredin the carbohydrate-extracts of indicated beverages. Mean values of fourpooled technical replicates were blotted to demonstrate extracted sugarconcentrations. Furthermore (D), the C6/C7 (sedoheptulose) ratio wasestablished by dividing the sum of glucose and fructose (C6) moleculesor weight by respective sedoheptulose (C7) unit (N.A.=not applicable).

FIG. 2: (A) Blot of normalized sedoheptulose kinase (CARKL) mRNAexpression levels in mouse liver over a period of 48 hrs was obtainedfrom the public available “Circadian Gene Expression Database—CIRCA”.Y-axis represents normalized gene expression and X-axis time in hours.(B and C) Reduced and oxidized nicotinamide adenine dinucleotide levelsof cells with perturbed CARKL expression were calculated from previouslyrecorded metabolomics data (Haschemi et al., 15 (2012), 813-826).RAW264.7 cell line either (B) expressing high CARKL levels byoverexpression or (C) low CARKL levels by shRNAmir expression werecompared to individual control cell lines to illustrate CARKL mediatednicotinamide adenine dinucleotide regulation. Data represent mean foldchange of three individual experiments +/−SEM. (D) CARKL mRNA levelswere measured in a mouse cDNA tissue library. Data represents normalized(to β-actin) mean CARKL expression relative to thymus CARKL expressionin fold change (all tissues n=3, S.D.).

FIG. 3: (A-E) Primary human adipocytes were obtained by differentiationof the stromal vascular cell fraction in the presence of glucose andfructose (GF, 1.5 g/L of each sugar; total carbohydrates 3 g/L) orsedoheptulose supplemented media, which was termed GFS (1 g/L of eachsugar; total carbohydrates 3 g/L), for eight days. Subsequently mRNAlevels of (A) CARKL, (B) adiponectin and (C) uncoupling protein 1(UCP-1) was assessed by RT-PCR and compared between the groups. (D)Cellular oxygen consumption rates (OCR) and (E) extracellularacidification rates (ECAR) of adipocytes were recorded in the presenceof indicated carbohydrate-mix (mean+/−S.D., n=9-11). (F) Tumor necrosisfactor alpha (TNFa) and (G) interleukin 6 (IL-6) mRNA expression levelsof mature murine adipocytes cultivated for three days in cell culturemedia containing either GF or GFS carbohydrate mix, or (H) IL-6expression in primary murine hepatocytes, as well as (I) TNFa and (J)IL-6 mRNA levels in primary bone marrow derived macrophages, both celltypes were pre-cultured for two days in GF or GFS media, were comparedbetween respective groups. Secretion of (K) TNFa and (L) IL-6 cytokinesover the period of two days was measured by ELISA in the supernatant ofbone marrow derived macrophages. LPS-induced (100 ng/ml) secretion of(M) TNFa 2 hrs after activation and (N) IL-6 6 hrs after activation inthe respective media by macrophages was also measured by ELISA. Datarepresents mean+/−S.D., n=3)

FIG. 4: In an in vitro kinase assay, which employs ADP formation asactivity readout, the sedoheptulose turnover rate at constant ATP (150μM) concentration was enhanced either by (A) increasing sedoheptuloseconcentration or (B) by increasing the amount of the rate-limitingenzyme sedoheptulose kinase (CARKL). (C-E) Hepatocytes from wilt-type(WT) and sedoheptulose kinase CARKL overexpressing mice werepre-cultured for two days in GFS containing media before basal (C) ECARand (D) OCR was recorded of these cells. (E) Hepatocytes from bothgenetic linages (CARKL and WT controls) were cultivated in GF or GFScontaining media and then pre-starved (no-carbohydrates) for 1 h tomeasure glucose-induced ECAR after the addition of glucose to reach afinal concentration of 1 g/L in the culture media mean+/−S.D., n=7-11).As metabolic in vivo indicators (F) the respiratory quotient (RQ) and(G) energy expenditure (EE, kcal/day/kĝ0.75) per activity by indirectcalorimetry were determined and compared CARKL to wild-type (WT)littermates during day (8 am to 4 pm) and night (8 pm to 4 am). Datarepresent mean values, n=3, +/−SD. (H) Insulin tolerance test in micewas performed on pre-starved (4 h) female CARKL-transgenic and wild typelittermate controls by i.p. injection of 0.75 U insulin/kg body weight.Blood glucose was measured before the injection (control) and 15, 30, 45and 60 minutes after injection and graphed as relative value in % ofcontrol. Data represent mean values+/−SD, n=3-4.

FIG. 5: (A) Lipid deposition in hepatocytes isolated from either WT orCARKL overexpressing mice and cultivated in control medium (GF) wasvisualized by oil red staining. Representative micrographs were acquiredwith a 20× objective. (B) Glucose-6-phosphate dehydrogenase activity wasmeasured in hepatocytes isolated from either WT or CARKL overexpressingmice and cultivated in control medium (GF) for two days. Arrows indicatere-localization of G6PD activity. Representative micrographs wereacquired with a 10× objective. (C and D) Osteoclasts from WT or CARKLoverexpressing mice were differentiated and cultivated for 7 days inCorning Osteo Assay plates in GF or GFS containing media. Theirresorption-activity was assessed by counting all pits per well whichwere visible at 20× magnification. Data represent mean values, n=2-3,+/−SD. (D) Two representative images of the largest resorption-pitsfound in WT osteoclast either cultured in GF or GFS containing media areshown. Micrographs were acquired by 40× magnification.

FIG. 6: (A) The immune system of sedoheptulose kinase overexpressingmice (CARKL) and control littermates was challenged with a sub-lethallipopolysaccharide injection (LPS, 7 mg/kg) and a panel of 19 cytokineswas measured 24 hrs after immune activation in serum. Data representsrelative mean fluorescence intensities (MFI) of individual cytokinesmeasured in wild type (WT=100%) and CARKL serum by milliplex map mousecytokine profiler (n=3, +/−SD). Monocyte chemoattractant protein-1(MCP-1) and TNFa mRNA expression of murine mature adipocytes isolatedfrom (B and C) subcutaneous or (D and E) epididymal white adipose tissueof WT and CARKL mice. Data represent mean values, n=3, +/−SD. (F)Incidence of stroke and rs465563 genotype. Data were compared byPearson's χ²-tests. ** . . . p<0.01. (G) Duration of initial stroke-freesurvival was assessed by Kaplan-Meier plots. Individuals homozygous forthe rs465563: [G]-allele show significantly shorter event-free timesthan carriers of the [A]-allele.

EXAMPLES Materials and Methods Carbohydrates

Glucose and fructose were purchased from Sigma Aldrich. Sedoheptulosewas isolated from the plant sedum Spectabile or sedum telephium(Haschemi et al., Cell Metab. 15 (2012), 813-826) and was furtherpurified by chromatography.

Glucose and Sedoheptulose Measurements in Carrots and Human Serum

Fresh carrots with an Austrian “organic-certificate” were sliced intosmall pieces and snap-frozen in liquid nitrogen. The same procedure wasapplied to leaves freshly harvested from indoor-grown Sedum Spectabileplants. Three samples of each tissue were pooled and homogenized topowder by glass-beads. The carbohydrate rich fractions of the sampleswere isolated by 20% H₂O and 80% MeOH extraction solution spiked with13C standards and processed for standard gas-chromatography coupled tomass spectrometry (GC-MS). Glucose and sedoheptulose were identified bytheir individual masses and their relative amount was derived from thepeak area of each individual analyte normalized to 13C content. Glucoseand sedoheptulose were also measured in equal volumes of serum fromovernight fasted or carrot fed individuals. The carrots were steamed andsome olive-oil, salt and pepper were used for flavour. Two hours beforeand two hours after the carrot containing meal human serum was preparedin VACUETTE® Z Serum Sep tubes from blood. Serum samples were processedas previously described and also analyzed by GC-MS as detailed above andin Ruiz-Matute A I et al., Chromatogr B Analyt Technol Biomed Life Sci.(2011) May 15 879(17-18).

Glucose, Fructose and Sedoheptulose Concentration in Beverages

Indicated beverages were purchased from a local grocery store and wereall pre-packed. From each beverage 1 ml (for each of four technicalreplicates) was aliquoted and vacuum concentrated on a SpeedVac. Thiswas followed by MeOH/H2O (80:20) precipitation and a centrifugation stepto clear the carbohydrate fraction in the supernatant from precipitate.Each supernatant was again lyophilized in a SpeedVac, rehydrated inequal volumes of water and technical replicates were pooled. Forcarbohydrate analysis a Dionex ICS-3000 DC metal-free system was usedwith a CarboPac PA1 column (250×4 mm) and a CarboPac PA1 guard column(both from Dionex) at a flow rate of 1 mL min-1. Elution was carried outisocratically with 16 mM NaOH for the first 20 min. Then a lineargradient to 100 mM NaOH was applied from 20 to 40 min followed by anincrease over two minutes to 200 mM hold until 47 min. The startingcondition with 16 mM NaOH was reached again at 49 min and kept until 70min. Parameters of the pulsed amperometric detection are exactly asrecommended in the Technical Note 21 (Optimal Settings for PulsedAmperometric Detection of Carbohydrates Using the Dionex ED40Electrochemical Detector) from Dionex. Chromatograms were evaluatedaccording to chromatograms of authentic glucose, fructose andsedoheptulose standards, each 100 μM.

Human Adipocyte Culture

Stromal vascular fraction cells (SVFs) from subcutanous white adiposetissue of a patient undergoing abdominal surgery were isolated asdescribed before (Lindroos et al, Cell Metab. (2013), July 2;18(1):62-74.). Briefly, adipose tissue was digested with Collagenase II(Worthington) and filtered. SVFs were separated from mature adipocytesby centrifugation, followed by the lysis of erythrocytes in theSVF-fraction (Buffer EL, Qiagen). The purified SVFs were cultured inDMEM/F12, 10% FBS (Gibco, Life Technologies). As soon as the cellsreached confluence, they were washed with DMEM/F12 w/o glucose (Biowest)and the medium was replaced by DMEM/F12 with a total carbohydrate loadof 3 g/L, containing 1.5 g/L glucose and fructose or 1 g/L each ofglucose, fructose, and sedoheptulose, respectively. Two days afterconfluence (day 0), differentiation was induced by adding 1 μMdexamethasone, 1.74 μM insulin, 5 μM troglitazone, 0.5 μM IBMX, 17 μMpantothenic acid and 33 μM biotin. After two days, IBMX anddexamethasone were omitted, while insulin and troglitazone were removedon day 4. 50% of the medium was replaced on day 6. Two days later, thecells were harvested for RNA isolation (RNeasy mini kit, Qiagen).

Mature Murine Adipocytes Ceiling Culture

Anterior and posterior subcutaneous adipose tissue and epididymal whiteadipose tissue was isolated from transgenic CARKL-mice and wild-typelittermate controls. Adipose tissue was digested as described above forthe human sample. After centrifugation, the layer of floating matureadipocytes was removed and re-centrifuged at 100 rcf for 10 minutes. 150μL of packed adipocytes were cultured under a floating coverslip insix-well plates, in DMEM/F12, 15% calf serum (PAA). 24 hours afterisolation, the wells were washed twice with PBS and the medium wasexchanged to DMEM/F12 with a total carbohydrate load of 3 g/L,containing 1.5 g/L glucose and fructose, or 1 g/L each of glucose,fructose and sedoheptulose, respectively. Three days later, the cellswere harvested for RNA isolation using TRIreagent (Sigma).

Hepatocyte Culture

Hepatocytes were isolated from male wild-type and CARKL overexpressingmice after the mice were sacrificed by hepatic in situ collagenaseperfusion. After perfusion, the liver was quickly removed, minced, andfiltered through a cell strainer (Fisher Scientific, Inc) into a 50 mlsterile tube. Hepatocytes in the resulting filtrate were purified fromthe nonparenchymal cells by centrifugation steps. The isolatedhepatocytes were seeded into plates at a concentration of 0.25×10̂6/ml inrespective DMEM-media containing 10% FBS and indicated carbohydratemixes at 3 g/L (glucose and fructose (GF), or glucose, fructose andsedoheptulose (GFS)) at least two days before experiments wereperformed.

Bone Marrow Derived Macrophages and Osteoclasts Culture

Mouse bones were isolated from male wild-type and CARKL overexpressingmice and bone marrow wash flushed, washed and cleared from redbloodcells by lysis, and subsequently differentiated in DMEM containing 25 mMglucose and 20 ng/ml M-CSF. After 4 days, cells for osteoclastdifferentiation were harvested and seeded in corning osteo assay plateswith differentiation media (see below), and for primary bone marrowderived macrophages (BMDM) the medium was renewed and furthersupplemented with M-SCF for two days. Differentiated BMDM were thenseeded in respective DMEM-media containing 10% FBS and indicatedcarbohydrate mixes at 3 g/L (glucose and fructose (GF), or glucose,fructose and sedoheptulose (GFS)) at least two days before experimentswere performed.

In Vitro Bone Resorption Assay

Osteoclast precursor cells (bone marrow cells treated with 20 ng/mlM-CSF for 4 days) were plated in 24-well Corning Osteo Assay plate inthe presence of 10 ng/ml M-CSF and 100 ng/ml RANKL in alpha-MEM mediumcontaining 10% FBS and 3 g/L either glucose and fructose (GF), orglucose, fructose, and sedoheptulose (GFS). The Medium was renewed everythird day. After 7 days, cells were removed with bleach and washed threetimes with water before Von Kossa staining according to standardprotocols from Corning (technical review) was performed to enhancecontrast between the intact assay surface and resorption pit. Each wellwas entirely photographed by TissueFax with an ×20 objective and allvisible resorption pits were counted and analysed blinded.

Extracellular Acidification Rates and Oxygen Consumption Rates

The XF-Analyzer (Seahorse Bio.) was used to measure changes in cellularmetabolism induced by different carbohydrates. Extracellularacidification rates (ECAR, mpH/min) and oxygen consumption rates (OCR,pmoles/min) were recorded in primary human adipocytes and primary murinehepatocytes according to the manufacture instructions. Briefly, cellswere seeded in seahorse cell plates at a density of 10̂4 cells per wellfor adipocytes and 0.5×10̂5 cells per well for hepatocytes. At the day ofexperiments the cells were washed with carbohydrate free media andincubated for one hour in indicated media containing carbohydrate mixesat a concentration of 3 g/L (glucose and fructose (GF), or glucose,fructose and sedoheptulose (GFS)). To measure glucose-induced ECAR,cells were starved for one hour in carbon free media before glucose (1g/L) was added to the well per automatic injection. Glucose-induced ECARwas normalized to respective ECAR before glucose addition (basal=100%).Changes induced by the different carbohydrate sources were expressed asrelative change in % to illustrate the effect of each carbon source.

Gene Expression Analysis

Briefly, total RNA was extracted and reverse-transcribed usingcommercial kits (QIAGEN, Applied Biosystems). Quantitative real-timePCRs were performed on an AbiPRISM 7900HT real-time cycler using iTaqSYBR Green Supermix with ROX (BioRad) to measure CARKL, adiponectin,UCP-1, TNFa, IL-6, MCP-1, and beta-actin expression as previouslydescribed (Haschemi et al., Cell Metab. 15 (2012), 813-826). Expressionof target gene was normalized to beta-actin expression. Relativeexpression of target gene was calculated by the delta-delta CT method.

Sedoheptulose Kinase Assay

The ADP Quest Assay (DiscoveRx) was used to indirectly measure S7Pformation by ADP accumulation over time according to the manufactureinstructions. Recombinant sedoheptulose kinase was previously producedin E. coli and purified by affinity purification (Haschemi et al., CellMetab. 15 (2012), 813-826).

Indirect Calorimetry

The respiratory quotient (VCO2 production/VO2 consumption) and energyexpenditure (EE, kcal/day/kĝ0.75) per activity of mice were measured inmetabolic cages (Harvard Apperatus) by indirect calorimetry. Either oneCARKL transgenic mouse or a wild-type littermate, was housed per cagefor one day before it was started to record mean respiratory quotientand energy expenditure per activity during day (8 am to 4 pm) and night(8 pm to 4 am).

Insulin Tolerance Test

Four hours pre-starved female CARKL-transgenic mice and wild typelittermate controls were i.p. injected with 0.75 U insulin/kg bodyweight. Blood glucose was measured before the injection and 15, 30, 45and 60 minutes after injection by using one-touch glucose strips (AccuCheck, Roche).

Oil Red Staining

Briefly, hepatocytes were cultured as indicated before the medium wasremoved and cells were fixed by 10% formalin. After a wash with 60%isopropanol the wells were incubated with Oil Red O solution(Sigma-Aldrich) and stained for 10 min, washed 4 times with water beforephotographs were taken at ×20 magnification.

Glucose-6-Phosphate Dehydrogenase Activity Assay

Cells were grown on polystyrene vessel tissue culture treated glassslides, snap frozen in liquid nitrogen and kept at −80° C. The enzymehistochemical procedure was based on the tetrazolium salt method asdescribed by Van Noorden and Frederiks. The incubation medium for thedemonstration of G6PD activity contained 18% (w/v) polyvinyl alcohol(average molecular weight 70,000-100,000) in 0.1 M Tris-Maleate buffer,pH 7.5, 15 mM glucose-6-phosphate, 0.8 mM NADP, 0.4 mM magnesiumchloride, 0.45 mM 1-methoxyphenazine methosulphate, 5 mM sodium azideand 5 mM tetranitroblue tetrazolium chloride. The medium was freshlyprepared immediately before incubation. Control reactions additionallycontained 40 mM glucosamine-6-phosphate. Cells were incubated at roomtemperature for 15 minutes under continuous mixing by orbital shaker.After incubation, cells were washed 3×5 minutes in 60° C. phosphatebuffered saline, dried, and mounted in glycerol gelatin, andphotographed within one day at ×10 magnification.

Immune Challenge and Cytokine Response Profiling In Vivo

For sublethal murine in vivo endotoxemia, 7 mg/kg LPS (Sigma Aldrich)were injected intraperitoneally in male CARKL transgenic or wild-typelittermates. Serum was isolated by VACUETTE® mini Z Serum Sep tubes fromwhole blood. Cytokine were measured by milliplex map mouse cytokineprofiler according to manufactures instructions (Millipore). All animalexperiments were carried out according to an ethical animal licenseprotocol and contract approved by the Medical University Vienna(BMWF-66.009/0140-II/10b/2010).

CARKL Single Nucleotide Polymorphism (SNP) Association-Study with Riskof Stroke.

Study Design: 578 neurologically asymptomatic patients from theInflammation and Carotid Artery—Risk for Atherosclerosis Study (ICARAS,Schillinger et al., (2005) Circulation 111 (17):2203-2209.)), recruiteduntil 03/2003, were included into the present analysis—inclusion andexclusion criteria have been published previously. Briefly, asymptomaticcarotid artery disease, representing the primary inclusion criterion,was defined as the absence of transient ischemic attacks (TIA),amaurosis fugax and stroke within the last 12 months or of residualsymptoms, respectively. Besides exhaustive medical examination(including anamnesis, physical status and blood testing), patientsunderwent sonography of the carotid arteries at baseline and at afollow-up 6-9 months after study inclusion as previously described(Schillinger et al., (2005) Circulation 111 (17):2203-2209.). Incidenceof cardiovascular events was recorded until 01/2006. This analysis wasapproved by the local ethics committee (EC-No. 1933/2012) and has beenperformed in accordance with the ethical standards specified by theDeclaration of Helsinki and its amendments.

Definitions: Arterial hypertension was defined as repetitive restingblood pressure above 140/90 mm Hg and was assumed to be present inpatients with antihypertensive medication. Hyperlipidemia was diagnosedin patients with a total serum cholesterol >200 mg/dL or LDLcholesterol >130 mg/dL and was considered to be present in all patientstaking lipid-lowering drugs. Diabetes mellitus was diagnosed as supposedby Expert Committee on the Diagnosis and Classification of DiabetesMellitus. Peripheral artery disease was graded using Fontaine'sclassification system, coronary artery disease was defined according tothe classification by the Canadian Cardiovascular Society (CCS).Anamnestic myocardial infarction was defined according to Alpert. Strokewas defined by a neurological deficit persisting >24 h and was evaluatedaccording to the modified Rankin stroke scale. Progression of carotidartery atherosclerosis was defined as an increase in stenosis by atleast one NASCET angiographic degree.

Genotyping: DNA was isolated from EDTA-anticoagulated whole blood bymeans of spin-column based nucleic acid purification. Genotyping wasdone on an ABI TaqMan® 7900HT fast-realtime thermocycler (AppliedBiosystems, Rotkreuz, Switzerland) using the 5′-Nuclease-Assay[7]. Thisassay includes the sequence-specific binding of labeled DNA-probes.During the annealing phase of each step, sequence-specific probescontaining a fluorophore on their 5′-ends bind their respective allele.The fluorescence of the fluorophore is masked by a 3′-quencher, untilthe 5′-Taq-polymerase separates these compounds by its 5′-exonucleaseactivity during the elongation phase. End-point-measurement of theintensity of sequence-specific fluorescence indicates the presence ofthe corresponding allele. In the present study, rs465563 was analyzed ina total reaction volume of 10 μL using a commercially available TaqMan®SNP genotyping assay (Assay ID: C_(—)717358_(—)1_, Applied Biosystems)and TaqMan® Genotyping Master Mix (Applied Biosystems) according to thestandard protocol supplied by the manufacturer. The results wereinterpreted using SDS 2.4 sequence detection software (AppliedBiosystems).

Statistical Analysis: Continuous data are given with respect to itsdistribution as mean and standard deviation or median and interquartilerange. Categorical data are presented as counts and percentage. Presenceof normal distribution within subgroups was assessed byKolmogorov-Smirnov-tests. Metric variables were compared by Mann-WhitneyU tests. Connections between genotypes and study end points as well asrelative risks were estimated by contingency-tables and Pearson'sχ²-tests. Event-free survival was estimated by Kaplan-Meier-Analysis,data was compared pairwise by Log Rank (Mantel-Cox) tests. Multivariablemodels were calculated by means of binary logistic regression andevaluated by drawing ROC (Receiver-Operator-Characteristics) plots.Results were considered statistically significant at p<0.05 unlessotherwise noted. All p-values were interpreted two-sided unlessotherwise stated. All statistical calculations were done using IBM SPSS20.0 (IBM Corporation, Armonk, USA).

Results Diet-Derived Sedoheptulose Uptake in Humans

To determine if nutritional sedoheptulose is absorbed from diets via thedigestive tract into the human blood, which is a critical step forsedoheptulose to become a metabolic active carbohydrate, glucose (C6)and sedoheptulose (C7) levels were measured in carrots and in humanserum. To determine the C6/C7 ratio of carrots plant tissue washomogenized, extracted the small molecule fractions and measuredsimultaneously relative glucose and sedoheptulose amounts by GC-MS (FIG.1A). Leaves of Sedum Spectabile, a succulent plant which contains highsedoheptulose levels, were analyzed in parallel as positive control. Theleaves of Sedum Spectabile contained glucose and approximately twice asmuch sedoheptulose, which resulted in a C6/C7 ratio of approximately0.5. In fresh food grade organic carrots almost equal quantities ofglucose and sedoheptulose were detected and thereby a C6/C7 ratio ofaround 1. Potatoes, in contrast to carrots, were reported to contain nosedoheptulose (Kardon et al., FEBS Lett. (2008) October 15, 582(23-24))and are therefore expected to possess a very high (or even infinite)C6/C7 ratio. Next it was addressed if diet-derived sedoheptulose,originating from carrots, reaches the human blood by monitoring serumlevel of glucose and sedoheptulose before and after a meal containingmainly steamed carrots (˜700 g per male adult) with some olive-oil, saltand pepper. Two hours after the meal, an approximately two-fold increasein sedoheptulose serum levels was observed in humans (FIG. 1B). Thisresult clearly indicated that nutritional sedoheptulose is absorbed bythe human digestive tract and that it enters the blood to becomeavailable as a carbohydrate for subsequent metabolism. Serum glucoselevels normalized already within two hours after food uptake and therebysuggest that humans metabolize sedoheptulose and glucose differently.

These results indicate that carrots, which are considered as healthyvegetables and constituents of diets around the world, are a naturalsedoheptulose source for humans. It is interesting to mention thatcarrots, although they contain carbohydrates such as glucose, aresuitable and well tolerated by diabetics and even protect people withcommon genetic risk factors for type-2 diabetes (Patel C J et al., HumGenet. 132 (2013): 495-508).

The C6/C7 Ratio in Natural and Manufactured Human Food Products

Next, the concentration of glucose, fructose and sedoheptulose wasmeasured in carbohydrate extracts from Coca-Cola®, Red Bull®, orange-,apple-, tomato-, and carrot-juice to establish the C6/C7 index forcommon beverages and thereby estimate the actual humansedoheptulose-exposure. Soft- and energy drinks contained glucose andfructose but no sedoheptulose (FIG. 1C). Fruit-juices contained similaramounts of C6 (the sum of glucose and fructose) as soft-/energy drinks(100 mM range), but in contrast to the latter they also containedsedoheptulose in the 100 μM range. In vegetable-juices glucose andfructose levels were reduced, whereas sedoheptulose concentrations werefound greatly increased (mM range). The C6/C7 ratios of the individualbeverages, calculated as molar—as well as weight ratios, are given inFIG. 1D. In two fruit-juices C6/C7 ratios of around 2000/1 were found.In tomato- and carrot-juices the ratio was 75/1 and 20/1, respectively;Coca-Cola® and Red Bull® contained no detectable amounts ofsedoheptulose. Hence, the C6/C7 ratio for these drinks could not becalculated and is therefore not applicable (N.A.) to such food productsif they are not additionally supplemented with C7. These findingsclearly indicate that manufactured food products lack carbohydratecomplexity and therefore also sedoheptulose. Notably, in absolutenumbers these data indicate that 1 kg of diet-derived sedoheptulose isconsumed per 9 kg diet-derived glucose if the natural carbon-mixture ofcarrot-juice was chosen over soft-/energy drinks—the consequences ofchronic sedoheptulose deprivation by an unbalanced human nutrition arecurrently unknown.

Sedoheptulose Kinase Oscillates and is Highly Expressed in MetabolicallyActive Tissue.

To understand how sedoheptulose kinase CARKL and therefore sedoheptulosemetabolism is distributed and controlled in various mammalian tissues,different tissues were tested for mRNA expression levels. CARKLexpression, in a publicly available database for circadian expressionprofiles (CircaDB), was found oscillating in mouse liver (period 20.0,FIG. 2A) and adrenal gland (period 24.0, data not shown).Sedoheptulose-7P levels were previously reported to oscillate in plants,peaking during the regenerative-phase of carbon-fixation to reconvertribose moieties. The mammalian circadian clock is in part set by theredox-state. CARKL overexpressing RAW264.7 cells indicated that highsedoheptulose kinase expression induces a 4 fold increase ofredox-factors like reduced nicotinamide, adenine dinucleotide (NADH) andalso glutathione (GSH) level increase (FIG. 2B and Haschemi et al., CellMetab. 15 (2012), 813-826). Deficiency of CARKL and therefore also lackof sedoheptulose turnover showed the opposite effect (FIG. 2C). Thisdata indicates that sedoheptulose metabolism is under the control ofcircadian rhythm and acts simultaneously as modulator of cellularredox-states. In order to specify which tissue consumes nutritionalsedoheptulose, mRNA levels of sedoheptulose kinase (CARKL) were measuredin a mouse cDNA tissue library (FIG. 2D). Sedoheptulose kinase is therate-limiting enzyme of sedoheptulose metabolism and was highlyexpressed in liver, kidney, in brown and white adipose tissue (BAT andWAT), digestive organs, in glands, in the male reproductive system aswell as in the brain. These results demonstrate that diet-derivedsedoheptulose can become a systemic carbohydrate source for many tissuesbut especially for metabolically active organs such as the liver, kidneyand adipose tissue.

Sedoheptulose Metabolism is Important for the Function and MetabolicHealth of Cells

To investigate the metabolic consequences of exposure to sedoheptulose,human and murine cells in the culture media were exposed to eitherglucose in combination with fructose (GF) or to glucose, fructose andsedoheptulose (GFS), while keeping the total carbohydrate load constant.The function of adipocytes, hepatocytes and macrophages was tested toevaluate the importance of sedoheptulose in complementing nutritionalcarbohydrate loads. The sedoheptulose used in these experiments wasisolated from sedum telephium plants.

In the human body, adipocytes fulfil the crucial function to storeenergy in form of lipids and are therefore very important cells tomaintain health. In metabolically unhealthy or in most morbidly obesepatients adipocytes do not function properly, increase their cell sizeto account for excessive lipid loads, become inflamed(meta-inflammation, a low-grade but chronic type of inflammation) andultimately contribute to the development of disorders like insulinresistance or cardio-vascular diseases. In human adipocytes,differentiated from subcutaneous precursor cells, an increase insedoheptulose kinase CARKL mRNA expression was found if sedoheptulosewas added to the culture media (FIG. 3A). Furthermore, an increase inadiponectin and UCP-1 expression, two functional relevant adipocytemarker genes, was observed in the GFS compared to GF treated cells(FIGS. 3B and 3C). To test for changes in cellular metabolism, cellularoxygen consumption rates (OCR) and extracellular acidification rates(ECAR) were measured as indicators for mitochondrial respiration andlactic acid fermentation, respectively. Human adipocytes, differentiatedand cultured in sedoheptulose containing media, showed comparable ECARto WT controls, while their cellular oxygen consumption rates wereincreased (FIGS. 3D and 3E).

Together, these data show that subcutaneous adipocytes maintain andimprove their function (increase in adiponectin, UCP-1 and oxygenconsumption) in the presence of sedoheptulose compared to a situationwhere only glucose and fructose are present as bio-fuel.

To better define the beneficial role of sedoheptulose in preventingmetabolically unhealthy states like chronic low-grade inflammation, thecytokine profiles of primary mature murine adipocytes, primary murinehepatocytes, and naïve as well as lipopolysaccharide (LPS)-inducedactivated primary macrophages cultured in GF or GFS media were measured.Mature adipocytes, which were cultivated in sedoheptulose containingmedia, produced less TNFa and IL-6 mRNA than adipocytes cultivated withglucose and fructose alone (FIGS. 3F and 3G). Similarly to that,hepatocytes also produced less IL-6 if cultivated with sedoheptulose(FIG. 3H). In primary macrophages basal TNFa and IL-6 mRNA expression,and cytokine secretion were found blocked by sedoheptulose containingmedia (FIG. 3I-L). LPS-induced macrophage activation resulted instrongly enhanced TNF-a and IL-6 secretion in GF cultured cells (FIGS.3M and 3N). LPS-induced macrophages cultivated in GFS media increasedIL-6 to comparable levels as GF treated macrophages, but reduced theirTNFa secretion by approximately 40%. This shows that addition ofsedoheptulose to macrophages on the one hand reduces their basalinflammatory state, measured by TNFa and IL-6, but on the other handretains their natural function in immunity as seen for IL-6.

To conclude, addition of sedoheptulose to the culture mediasignificantly enhanced subcutaneous adipocyte function and reduced theinflammatory state of cells and thereby might positively influence thepathogenesis of obesity, type II diabetes and cardiovascular diseasesalso summarized in part by the metabolic syndrome.

Sedoheptulose Kinase CARKL Overexpression

Transgenic mice overexpressing sedoheptulose kinase ubiquitously in theentire body were generated to isolate genetically engineered primarycells overexpressing CARKL and to test sedoheptulose metabolism in vivo.CARKL transgenic mice did not show any obvious phenotypes and theirorgan weight was not different to organ weights of WT littermate controlanimals (data not shown). It was hypothesized that increasedsedoheptulose metabolism can either be achieved by increasing theconcentration of the substrate sedoheptulose, or by elevating the levelof its rate-limiting enzyme sedoheptulose kinase (CARKL). To test thisin an in vitro kinase assay, a constant amount of recombinant CARKLprotein was incubated with increased doses of sedoheptulose. Thisresulted in enhanced sedoheptulose turnover rates as measured by ADPformation (FIG. 4A). The same effect was observed by increasing CARKLprotein concentration, while sedoheptulose concentration was keptconstant (FIG. 4B). These results demonstrate that CARKL overexpressionis a valid model to investigate the effects of increased sedoheptulosemetabolism. Next, primary hepatocytes, as liver tissue appeared toendogenously metabolize sedoheptulose (FIG. 2D), were isolated to studythe effects of C7- and C6-metabolism at increased CARKL expressionlevels.

Hepatocytes were cultured in media with a C6/C7 sugar ratio of 2:1 andmetabolically assessed by comparing the ECAR and OCR as more generalmetabolic-parameters. Hepatocytes from CARKL overexpressing micesignificantly increased their metabolic rate as increased ECAR and OCRlevels indicated if compared to WT control cells (FIGS. 4C and 4D). Itappeared that sedoheptulose induced a more efficient metabolism in thesecells. To test for the relevance of sedoheptulose and sedoheptulosekinase expression together, hepatocytes isolated from WT and CARKL micewere incubated in GF and GFS containing culture media and theirglucose-induced ECAR was compared (after 1 hr carbohydrate starvation)as a measure to control cellular glycolysis. In WT hepatocytesglucose-induced ECAR was reduced by sedoheptulose administration (FIG.4E). In line with this data, glucose-stimulated hepatocytes from WTanimals cultured in GF (without sedoheptulose) routed a larger part ofglucose to lactic acid fermentation, whereas cells from CARKLoverexpressing mice appeared to balance the same glucose bolus moreefficiently.

These results further supported the previous data and clearly pointedout that increased sedoheptulose metabolism has distinct and beneficialeffects on cells, which are important for metabolic health.

Sedoheptulose Metabolism In Vivo

After validating cellular phenotypes of sedoheptulose kinaseoverexpression in hepatocytes the metabolic effect of increasedsedoheptulose turnover to an entire organism was investigated bycomparing the respiratory quotient (RQ; RQ=exhaled CO₂/oxygenconsumption), as well as energy expenditure per physical activity ofcontrol and sedoheptulose kinase transgenic mice, measured by indirectcalorimetry during day and night as systemic indicators of metabolism(FIGS. 4F and 4G). Sedoheptulose kinase overexpressing animals (CARKL)have lower RQ values compared to wild type (WT) littermates. CARKL miceshowed increased oxygen consumption and therefore lower RQ values, andthereby partly mirrored the effects of sedoheptulose on cells.Furthermore, CARKL mice also tend to have a lower EE per activity ifcompared to control mice. The insulin sensitivity of female CARKLtransgenic and WT mice were also compared by an insulin tolerance test(ITT), to investigate if sedoheptulose metabolism also affects insulinsignalling in vivo. Insulin injection (i.p.) lowered the blood glucoselevel in mice overexpressing sedoheptulose kinase faster than in WTcontrols, indicating that insulin sensitivity was also increased inthese animals by increasing sedoheptulose metabolism (FIG. 4H). In thiscontext it is important to mention that induction of insulin resistancein mature adipocytes, by TNFa and hypoxia treatment, resulted insedoheptulose kinase loss (public Gene expression omnibus data availableat http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM1261655).

Together, these experiments showed that increased sedoheptulosemetabolism does not only alter metabolism in vitro but also in vivo. Thelower RQ values observed in CARKL mice combined with the by trend lowerEE per physical activity and the increased insulin sensitivity areindicative for a more efficient and sustainable metabolic program, whichcan be induced by increasing sedoheptulose metabolism.

Hepatic Lipid Deposition is Reduced by CARKL

Extensive lipid deposition in hepatocytes results in fatty liver diseaseand can be accompanied by inflammation (i.e. non-alcoholicsteatohepatitis (NASH)) causing liver fibrosis, cirrhosis, andeventually hepatocellular carcinoma. The lipid content of hepatocytesfrom WT and CARKL overexpressing mice was compared and it was found thathepatocytes from CARKL mice contained less lipid droplets than their WTcontrols (FIG. 5A).

In conclusion, high liver fat content, especially in combination withinflammation, including malfunction of adipocytes, increases risk ofmany diseases including type 2 diabetes or cancer, thus both, thereduction of inflammation and lipid accumulation in hepatocytes bysedoheptulose might be especially valuable for human nutrition (seevegetables, which contain high C7 levels).

CARKL Regulates Glucose-6-phosphate Dehydrogenase Activity.

Glucose-6-phosphate dehydrogenase (G6PD), the rate limiting enzyme ofthe oxidative arm of the pentose phosphate pathway (PPP) which generatesC5 bodies i.e. for nucleotide synthesis and redox-equivalents, waspreviously shown to be directly inhibited by p53 (tumor-suppressor)(Jiang et al., Nat Cell Biol. (2011) March; 13(3):310-6). Mutation inp53, the most frequently mutated gene in human tumors, results inhyper-activation of G6PD and lipid accumulation in hepatocytes.Sedoheptulose kinase is the rate-limiting kinase of the non-oxidativearm of the PPP and was previously shown to reduce the oxidativePPP-flux, at least in macrophages. To test for a possible regulation ofG6PD activity in hepatocytes by sedoheptulose metabolism, the enzymeactivity of WT and CARKL overexpressing cells was compared (FIG. 5B). InWT control cells G6PD activity was observed to be evenly distributed,whereas in cells from CARKL mice a re-localization of G6PD and thereby areduced activity in the inner space of the cells was observed.Interestingly, the same phenotype was achieved by incubating WT cells incell culture media with sedoheptulose (not shown). If re-localizationand/or inhibition of G6PD by sedoheptulose kinase overexpression andsedoheptulose incubation were the cause for the reduced lipidaccumulation in hepatocytes (FIG. 4A) remains to be established.Nevertheless, this data in combination with data from macrophages showsthat an elevated sedoheptulose level may act as a countermeasure for p53loss in cancer cells, at least by negatively regulating G6PD activity insome instances. Another example are estrogen dependent breast cancercells which decrease CARKL expression in response to growth factorestrogen (public GeneChip repository data available athttp://www.ncbi.nlm.nih.gov/geoprofiles/; ID: 53372898;GDS3285/219713_at/SHPK), which in turn might again result in increasedG6PD activity and subsequently tumor growth.

This data also implements sedoheptulose metabolism as a suitable targetto regulate G6DP activity and for future anti-cancer strategies.

Sedoheptulose and Bone Metabolism

Bone is an extremely dynamic tissue. The fine balance of bone formationand resorption is essential for the proper mechanical stability of bone.Disequilibrium between bone formation and resorption is the cause ofosteoporosis, a highly prevalent condition of reduced bone density anddisturbed bone micro-architecture resulting in bone fragility andpathological fractures. As the osteoclasts, the cells responsible forbone resorption are derived from the monocyte lineage, whose function isstrongly impacted by sedoheptulose metabolism, it was also testedwhether osteoclast function may be modulated by the C6/C7 ratio.

Bone marrow cells were differentiated in vitro to osteoclasts byexposing the cells to M-CSF in combination with RANKL. The cells werecultured in plates, pre-coated with a bone mineral matrix at the bottomof the well, to provide substrate for their bone-resorption activity.Analysis of the substrate resorption clearly demonstrated that both,CARKL overexpression as well as increasing C7 content in the culturemedia, reduced pit formation by osteoclasts (FIG. 5C). Interestingly,larger resorption-pits were found only in the absence of sedoheptulose(FIG. 5D).

Again, this data supports the concept of the present invention that awell-balanced C6/C7 ratio is crucial to maintain health and that C7 hasthe potential to alleviate the pathophysiologic situations of i.e.osteoporosis in humans.

Sedoheptulose Kinase Regulates the Immune Response of Animals

Data of CARKL overexpression and its effects on the mouse immune-systemin a model of acute immune activation and in low-grade inflammation ofmature adipose tissue is presented with the present examples. In CARKLmice acute-inflammation was elicited by a sub-lethal lipopolysaccharide(LPS) injection, which resulted in a suppressed cytokine responsecompared to wild-type controls (FIG. 6A). A panel of 19 cytokines wasmeasured in WT and CARKL mouse serum, which was isolated 24 hrs afterLPS injection (7 mg/kg, IP), by mouse cytokine profiler. Twelvecytokines reached the threshold of 50% mean change, which was defined ascut-off to highlight cytokines regulated by CARKL overexpression invivo. IL-13, MIP1α, RNATES, IL-12 (p70), IL-2, TNF-α, MCP1, KC, GM-CSF,IL-6, IL-17 and IL-15 were decreased by at least 50% mean change inCARKL mice compared to wild-type littermates. Cytokines IL-6, IL-17 andIL-15 even reached a threshold of 75% mean change. In addition toacute-inflammation, the basal levels of pro-inflammatory cytokines inmature adipose tissue were also assessed from two different depots(subcutaneous and epididymal adipose tissue) of CARKL and WT controlmice. Consistently with the reduction in LPS-induced inflammation, mRNAlevels of TNFa and MCP1, a primary adipokine which recruits macrophagesto adipose tissue and which belongs to the pro-inflammatory cytokines,were found blunted by CARKL overexpression (FIG. 6B-E).

This indicates that a nutritional supplement containing sedoheptulosemight dampen acute and low-grade chronic inflammation. Therefore,targeting C7 metabolism, either by nutritional or clinically appliedsedoheptulose appears as an effective measure to reduce inflammation andassociated disorders.

A single nucleotide polymorphism (SNP) in the 3′-UTR of the CARKL geneis associated with the risk of stroke.

535 neurologically asymptomatic patients from the Inflammation andCarotid Artery—Risk for Atherosclerosis Study (ICARAS, Schillinger etal., (2005) Circulation 111 (17):2203-2209) were genotyped for thers465563 [A] to [G] substitution in the 3′-UTR of the CARKL gene. 33(6.2%) patients suffered a stroke within the observation period. Inthose patients, the distribution of rs465563 alleles was significantlydifferent compared to the allele frequencies of the rest of the studypopulation (χ²=12.639, df=2, p=0.002, see FIG. 6F). In detail,homozygous carriers of the [G]-allele bore a relative risk of 3.93 (95%CI: 1.72-9.01) compared to [A]-homozygous, and 3.10 (95% CI: 1.40-6.87)compared to heterozygous individuals, respectively. Statisticalindependence of rs465563 genotype information was assessed by a binarylogistic regression model providing age, sex, histories of myocardialinfarction and stroke, nicotine consumption, body mass index, as well ascertain comorbidities (hyperlipidemia, hypertension, diabetes mellitus)as covariates (model: χ²=30.476, df=11, p=0.001). Within this model,carrier status of the [G];[G] genotype presented as significantpredictor (odds ratio=5.015, 95% CI: 1.803-13.943) when compared topatients homozygous for the [A]-allele. Moreover, it has been assessedby drawing Kaplan-Meier plots whether initial stroke-free survival timedepends on rs465563 carrier status. Indeed, homozygous carriers of the[G]-allele (1518.3 days, 95% CI: 1419.2-1617.4) presented withsignificantly shorter event-free survival than heterozygous individuals(1619.1 days, 95% CI: 1584.8-1653.3, p=0.005) and carriers of the[A];[A]-genotype (1788.1 days, 95% CI: 1755.7-1820.4, p=4.9·10−4) (FIG.6G). Individuals homozygous for the rs465563: [G]-allele showsignificantly shorter event-free times than carriers of the [A]-allele.

The mechanistical background of this clinical association remains to beclarified. However, given the ability of CARKL to modulate macrophagepolarization and the key role of the latter in the formation andprogression of atherosclerotic plaques, it seems likely that thers465563 polymorphism may impact on the risk of cerebrovascular eventsvia modulation of macrophage polarization within atheroscleroticplaques. This notion is supported by the observation according to thepresent invention that the 3′-UTR of the CARKL gene is mainlytranscribed in macrophages.

In conclusion, this association and the provided data on metabolism andfunction of adipocytes, hepatocytes, and macrophages in combination withreduced inflammation and enhanced insulin sensitivity in vitro and invivo strongly indicates that sedoheptulose (C7-sugar) is a criticalconstituent for human nutrition and simultaneously a highly relevantmedication to treat i.e. C7-defiencies or other metabolic unhealthydisease states.

1.-16. (canceled)
 17. A method of treating inflammation comprising:obtaining sedoheptulose; and administering said sedheptulose to asubject; wherein inflammation is treated in the subject.
 18. The methodof claim 17, wherein the inflammation is a chronic inflammation.
 19. Themethod of claim 17, wherein the inflammation is a systemic inflammatoryresponse syndrome (SIRS) or sepsis.
 20. The method of claim 19, whereinthe inflammation is further defined as massive systemic inflammation.21. The method of claim 20, wherein the massive systemic inflammation isin response to polytrauma, in response to infection, includesorgan-specific manifestations, acute respiratory distress syndrome(ARDS) and/or multiple organ dysfunction syndrome (MODS).
 22. The methodof claim 17, wherein the inflammation is meta-inflammation orinflammation defined as chronic (low grade) systemic inflammation asreflected by increased levels of pro-inflammatory cytokines or otherinflammation markers in peripheral blood and involved in aging,adiposity, atherosclerosis, impaired glucose tolerance and type 2diabetes, neurodegenerative disorders, major depressive disorder (MDD),or cancer.
 23. The method of claim 22, wherein chronic (low grade)systemic inflammation is reflected by increased levels of tumor necrosisfactor alpha, interleukin-6, c-reactive protein, or serum amyloid alpha.24. The method of claim 17, wherein the inflammation is involved,causally or otherwise, in the pathogenesis of an inflammation-relateddisorder, impaired glucose tolerance, diabetes mellitus and diabeticcomplications, metabolic syndrome, atherosclerosis, inflammatory boweldisease, colitis ulcerosa and rarer entities, chronic inflammatory liverdisease including chronic viral hepatitis, autoimmune hepatitis, primarysclerosing cholangitis and fatty liver disease in all stages, chronicobstructive pulmonary disease (COPD) and asthma bronciale, inflammatorydiseases of the central nervous system, Alzheimer's and Parkinson'sdisease, or peripheral nervous system, Guillain-Barre and relatedsyndromes, and neuritides/neuropathies associated with autoimmunecollagen tissue diseases or chronic infection, osteoporosis,osteoarthritis, arthritides, and other rheumatic disorders, vasculitisof any etiology and manifestation, age-related macular degeneration(AMD) chronic inflammatory skin disorders, periodontitis, majordepressive disorder, or cancer.
 25. The method of claim 24, wherein theinflammation is involved, causally or otherwise, in the pathogenesis ofadiposity, Crohn's disease, multiple sclerosis, diabetic polyneuropathy,Lyme disease, rheumatoid arthritis, ankylosing spondylitis, autoimmuneconnective tissue disorders, myositis, psoriasis, or atopic dermatitis.26. The method of claim 17, wherein the inflammation is involved in thepathogenesis of cancer comprising cancer causation, progression,metastasis, resistance to therapy, relapse after therapy, or a cancermanifestation.
 27. The method of claim 26, wherein the cancermanifestation comprises cancer-related cachexia, anemia, fatigue, ordepression.
 28. The method of claim 17, wherein the inflammation isinvolved in acute or chronic transplant rejection or in acute or chronicgraft-versus-host disease following autologous, syngeneic or allogeneicbone marrow, peripheral blood stem cell (PBSC) or cord bloodtransplantation.
 29. The method of claim 28, wherein the inflammation isinvolved in acute or chronic transplant rejection of an allogeneic lung,liver, kidney, heart, intestine, or pancreatic islet cell transplant.30. The method of claim 17, wherein the inflammation is selected fromthe group consisting of an inflammation of adipose tissue, aninflammation of the nervous system, an inflammation of blood vessels andan inflammation of the peritoneum.
 31. The method of claim 30, where theinflammation of the peritoneum is a result of peritoneal dialysis. 32.The method of claim 17, wherein the sedoheptulose is administered in anamount of 0.001 to 300 g per daily dosage.
 33. A method for storage oforgans for transplantation purposes wherein the organ to be transplantedto a patient is stored in a storage solution comprising sedoheptulose.34. The method of claim 33, wherein the storage solution comprisessedoheptulose in a concentration of 0.001 to 500 mg/ml, defined as theconcentration of all liquid material attached to or covering the organto be transplanted.
 35. A peritoneal dialysis solution containingsedoheptulose in a concentration of 0.001 to 500 mg/ml.
 36. Theperitoneal dialysis solution of claim 35, further defined as containingsedoheptulose in a concentration of 0.01 to 300 mg/ml.